It is our hypothesis that malignant transformation of human endometrial stromal cells and epithelial cells involves a multistep process, characterized by a sequence of genetic alteration that is, in whole or part, unique to this tissue. The goal of this project is to identify steps in the process of malignant transformation of these human cells. Building on our recent success in culture of normal human endometrial epithelial cells, we propose to transform these cells by repetitive treatments with MNNG and/or transfection of oncogenes. We seek to determine if similar steps are involved in transformation of epithelial and stromal cells. Endometrial stromal cells treated with chemical carcinogens reached partial transformation whereas transfections of oncogenes produced full transformation. We will use combinations of oncogene transfection and carcinogen treatment to transform these cells to identify which genetic alteration is not efficiently produced by chemical carcinogens. We also propose to transform human endometrial stromal cells by transfection of oncogenes that can be externally regulated at the genetic and/or protein product level. This will provide cells of identical genotype that can be made to express phenotypes from normal to fully transformed. By altering the activity of single transfected oncogenes, we may learn their roles in transformation and determine if these are quantitative effects based on their level of expression. These cells will provide an exceptional system for subtractive cDNA library analysis of altered gene expression in transformation. Transfection of neomycin resistance gene regulated by RSV promoter (pRSVneo) causes some of the steps in the progression to malignant transformation. pRSVneo co-transfected with c-myc confers full transformation. It is our hypothesis that RSV promoter is abnormally activating a gene located cis to its insertion site in the host cell genome. With RSV promoter as a genetic marker, we will seek genes that are activated by it as potential natural proto-oncogenes for human endometrium. Mesenchymal growth factor genes are expressed in endometrial cells after stimulation by macrophages or phorbol ester tumor promoters. We will determine whether these genes are abnormally regulated in transformed endometrial cells and if they are proto-oncogenes for this cell type.